The specific aims of this proposal are to identify the nucleotide sequences which are responsible for regulation in vivo of two early adenovirus type 5 (Ad5) transcription units, regions E1B and E4. Regions E1B and E4 are transcriptionally inactive immediately after infection with Ad5. Expression of a pre-early viral transcription unit (region E1A) results in activation of the E1B and E4 transcription units. At later times in infection, transcription of region E4 is specifically repressed by a product from early region 2 (E2). I propose to introduce targeted deletions in the 5 feet flanking and 5 feet noncoding sequences of Ad5 early regions E1B and E4 and analyze the effects of these mutations on transcriptional control in vivo. Large, and subsequently small, deletions will be targeted in these sequences in vitro using plasmids containing Ad5 DNA. The mutations will then be rebuilt into intact virus. Viruses containing mutations will be used to infect HeLa cells in culture and rates of nuclear transcription as well as cytoplasmic levels of E1B and E4 transcripts in vivo will be analyzed. These analyses will define at the nucleotide level the sequences which are responsible for activation of transcription of regions E1B and E4 by the products of region E1A. Also, the mutational analysis of the E4 transcriptional control region will more clearly define the mechanism(s) involved in the specific repression of E4 transcription by an E2 gene product. A key step in oncogenic transformation, presumably, is the perturbation of cellular control mechanisms which regulate cell growth. A potentially critical regulatory mechanism is the positive and negative control of transcription. The long term objective of my studies is to understand control of eukaryotic gene expression using virus-host models as a probe. Ad5 provides us with an extremely useful system to study the intricate and elaborate regulatory mechanisms which control viral gene expression. The delineation of transcriptional regulatory sequences in the Ad5 genome is a key step in understanding the control of viral, and ultimately cellular, gene expression.